Chronic itch is a presenting sign in numerous diseases associated with the skin, the immune, and the nervous systems. Chronic itch is largely resistant to conventional antihistamines, and few effective therapies are available. In order to develop novel therapeutics against chronic itch, there is a pressing need for identification of itc-specific signaling molecules involved. We have recently uncovered a small subset of itch-specific neurons which express gastrin-releasing peptide receptor (GRPR), an itch receptor, in the spinal cord. GRPR+ neurons are required for pruritoceptive but not for nociceptive transmission, indicating that they contain a myriad of itch-specific genes. To facilitate the identification of novel itch genes in GRPR+ neurons, we design a novel strategy that combines a genome-wide profiling of gene expression in GRPR+ neurons with functional screening, including behavioral tests and Ca2+ imaging. Gene expression profiles between the superficial laminae with normal GRPR+ neurons and without are compared. This strategy enables us to search for the genes that are expressed in GRPR+ neurons and are functionally required for mediating nonhistaminergic itch. Three sets of experiments will be performed to test the key hypothesis that there are distinct signaling signatures for histaminergic and nonhistaminergic itch in the spinal cord. First, genome-wide profiling of gene expression in GRPR+ neurons by microarray, qRT-PCR and in situ hybridization studies will be performed. The candidate genes expressed in GRPR+ neurons will be identified. Second, the involvement of the candidate genes in acute and chronic itch will be evaluated using a siRNA knockdown approach, and the key genes for mediating nonhistaminergic itch in acute or chronic itch will be identified. Third, the relationship between the candidate genes and GRPR will be determined by examining whether the knockdown of the gene would impair intrathecal GRP-evoked scratching and would further impact scratching behavior of GRPR mutant mice. In addition, whether the candidate genes are involved in GRPR-dependent Ca2+ signaling will be examined in a heterologous system using siRNA knockdown and Ca2+ imaging. The proposed studies are highly translational because the itch mediators are identified directly from chronic itch models, and likely to be itch-specific. Our proposed studies will significantly expand the pool of the targets that may be explored for future drug discovery program.